Numerous examples of cell-cell heterogeneity have been identified in immunology, including the myeloid cytokine response to microbial stimuli and the full extent of this diversity is not yet known. Standard assays conceal heterogeneity by averaging the responses of a population of cells, and thus obscure the underlying regulatory mechanisms. In this R21 Exploratory/Developmental Research Grant, we propose to quantitatively characterize macrophage diversity by building a microfluidic device to measure temporal cytokine profiles from multiple, individually isolated, live cells. We will adapt and integrate recent developments in scalable microfluidics with a multiplexed immunoassay technology to measure the diversity of macrophage responses at the single-cell level. The device will isolate single macrophage cells in ~0.25 nl volumes, and detect multiple secreted proteins using antibody-coated, color-coded microspheres surrounding the cells. By assaying single live cells directly in the device, our approach is distinct from other microfluidic efforts that simply offer miniaturized assays of samples derive from populations of cells. This technology is sufficiently scalable to assay hundreds of cells in parallel to quantitatively characterize cell-cell heterogeneity. Our bead-based approach permits multiple responses to be measured non-destructively from single cells, and thus has the potential to derive their temporal, co-ordinate response patterns and thereby disentangle the interwoven elements regulating cytokine secretion. We have identified the critical hurdles to our approach, and our preliminary data demonstrate solutions to each of these challenges. Our specific aims define milestones in the development of a practical device. We will demonstrate all of the elements required to eventually scale the technology to the level of monitoring 20 different responses, at multiple time points, from each of several hundred cells, with a single device. We anticipate that this device will be useful for defining co-ordinate regulation of complex processes, and will find broad utility in many areas of biology.